A polymeric substrate having a glass-like surface and a chip made of said polymeric
substrate
The present invention relates to a polymeric substrate having a glass-like and /or an etchedglass-
like surface and to a chip made of at least one such polymeric substrate. The present
invention also relates to a method of providing a polymeric substrate with a glass-like and /or
an etched-glass-like surface. Moreover, the present invention relates to a kit for manufactur¬
ing a chip using such polymeric substrate. Moreover, the present invention relates to the use
of a polymeric substrate having a glass-like surface for manufacturing a chip.
The integration of many analytical operations on a minaturized platform has been described
as the "lab-on-chip" concept by Manz and Widmer already in the last decade (D.J. Harrison,
A. Manz, Z. Fan, H. Luedi, H.M. Widmer, Anal. Chem. 64, 1992, 1926). Such microchip
systems including microfluidic systems, sensors, arrays (so-called biochips), chemical
synthesis on-chip, to name a few, allow small sample volume and low power consumption,
enable sample treatment, various chemical reactions, fast separation and detection times.
Since the concept emerged, the field grew rapidly and the developments regarding the
applications to new analytical areas and novel materials have been summarized in some
reviews. (D.R. Reyes, D. Iossifidis, P.A. Auroux, A. Manz, Anal. Chem. 74, 2002, 2623; P.A.
Auroux, D. Iossifidis, D.R. Reyes, A. Manz, Anal. Chem. 74, 2002, 2637).
These microsystem chips or microfluidic chips or microchips are mostly prepared from glass,
silicon or fused silica substrate materials due to their good optical properties (transparent in
the 450-700 nm range, low auto-fluorescence) and chemical inertness. However, devices
based on these materials are costly because they are fabricated using semiconductor microfabrication
techniques that include the formation of etching mask, photolithography, and sub¬
strate etching. Therefore, alternative materials have been investigated and especially various
polymers as alternative materials, such as as polydimethylsiloxane (PDMS), poly(methyl
methacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), polyethyleneterephthalate
(PETG) , polyvinylchloride (PVC) and polyimide (PI)„ have been studied because of their
lower cost, compatibility with biomolecules, optical transparency, number of replication
strategies and disposability (H. Becker, L.E. Locascio Gartner, Talanta, 56, 2002, 267).
Flow of fluids in macroscopic channels is generally determined by properties intrinsic to the
fluid like, e.g., its viscosity. In miniaturized systems, such as microfluidic chips having
miniaturized capillaries but also high-throughput array formats, surface properties and surface
effects play a key role because of the large surface-to-volume ratio when compared to
classical macroscopic systems and the fact that interactions of fluids with materials are
determined by the surface, and not the bulk properties of these materials. For good
performance of such a chip one of the main issues is therefore a well defined and
homogeneous channel geometry and surface property.
One of the major obstacles when changing the substrate material to plastic is the difference in
the surface properties of the new substrate materials and resulting differences in the
interaction with the analyte. For example, a common problem of most microfluidic devices
fabricated from polymeric material is the adsorption of the analytes to the channel walls
during the separation process, especially of molecules such as proteins or of surfactants such
as sodium dodecyl sulfate (SDS) used in protein separation gels with their hydrophobic side
facing the hydrophobic polymer surface. This can result not only in sample loss, but also in
changes in electroosmotic flow or analyte-wall interaction leading to deterioration of
separation performance. For other analytical microchip systems, such as enzyme reactors or
bioassay platforms, an efficient binding of reagents and bioprobes is essential. Another
problem is the separation of components of the analyte matrix from the channel surface,
leading to a layer of enhanced electroosmotic flow, which may disturb the electrophoretic
measurements.
Thus, surface modification and treatment of the polymeric materials is performed to control
non-specific adsorption of bioanalytes on the walls. Methods for the surface modification
include dynamic coating (non-permanent) and covalently (chemically) bound permanent
coatings (J.Liu, M.L. Lee, Electrophoresis, 2006, 27, 3533-3546; D. Belder, M. Ludwig,
Electrophoresis, 2003, 24, 3595-3606). Reported are also surface modifications of polymeric
substrates by employing plasma, electron beam or ion beam. Those modifications are
regarded as permanent but some properties are, not stable over time like e.g., the
hydrophilicity.
Dynamic coating is a convenient modification to perform. In this case, surface-active coating
materials or surface modifiers are brought in contact with the surface, for example by rinsing
through the channel. The coating materials are physisorbed onto the surface. However, the
potential of dynamic surface modification is limited in many application due to eventual
desorption from the polymer surface resulting again in surface property change during usage.
Further the desorbed surface modifiers could also interact with the analytes.
Permanent surface modification is therefore regarded as the most effective way for surface
modification. Ideally, the modification is stable and does not have to be regenerated.
However, since the modification needs specific chemical reactions or treatments it is often
more labor-intensive in the production process.
Assays are also a biotechnological application where polymer material can be used for the
device preparation. Transferring assay chemistries which have been developed for glass often
requires the addition of detergents to ensure the wetting of the hydrophobic plastic surface or
to avoid the sticking of proteins or other biomolecules on these hydrophobic surfaces. Adding
such detergents may negatively impact the performance of the assay, since such substances
can lead to denaturation of proteins or other biomolecules. Large protein molecules can easily
lose their functionality in the presence of detergents or other surface active substances. It
should also be mentioned that Cells or cell fragments are most easily damaged or destroyed
by exposure to detergents or hydrophobic plastic surfaces. Any of the above examples repre¬
sent cases, where the protein or biomolecule, large protein assemblies or cells and cell frag¬
ments are exposed to none native conditions in which they may behave very differently from
within their natural environment.
Accordingly, there is a need for materials that allow easy and low cost fabrication of systems
for biotechnological application whilst at the same time providing the advantageous
properties of glass-substrates. Accordingly, it was an object of the present invention to
provide means for the efficient fabrication of chips for various applications, in particular
biotechnological applications. It was also an object of the present invention to provide means
for the efficient fabrication of assay systems, in particular for biotechnological applications.
This object is solved by a polymeric substrate having a glass-like surface, in particular an
etched-glass-like surface, said glass-like surface, in particular said etched-glass-Iike surface
mimicking the surface of glass, in particular etched-glass in one or several of:
chemical content, chemical composition, chemical structure, homogeneity, roughness, mor¬
phology, in particular porosity, hydrophilicity, surface energy and adsorption affinity, surface
functionality, chemical and physical surface reactivity, zeta potential and surface charge.
In one embodiment, said surface has been modified by plasma treatment and/or reactive ion
treatment for increased roughness and hydrophilicity and/or is a thin film of silicon oxide
and/or is a polymer thin film having increased intrinsic roughness and/or intrinsic porosity
and/or increased hydrophilicity.
In one embodiment, said polymeric substrate is a polymer of synthetic or natural origin, pref¬
erably injection-moldable polymers, more preferably one of the material classes containing:
polyolefines, polyethers, polyesters, polyamides, polyimides, polyvinylchlorides, polyacrylates;
including their modifications, derivates, derivatives and copolymers; more specifically
one of the list containing acrylnitril-butadien-styrole (ABS), cyclo-olefin-polymers and co¬
polymers (COC/COP), Polymethylene-methacrylate (PMMA), Polycarbonate (PC), Polystyrole
(PS), Polypropylene (PP), Polyvinylchloride (PVC), Polyamide (PA), Polyethylene (PE),
Polyethylene-terephthalate (PET), Polytetrafluor-ethylene (PTFE), Polyoxymethylene (POM),
Thermoplastic elastomers (TPE), thermoplastic polyurethane (TPU), Polyimide (PI), Polyether-
ether-ketone (PEEK), Polylactic acid (PLA), polymethylpentene (PMP), and derivatives
thereof, wherein the polymer is optionally filled with an inorganic material such as carbon
black, oxides such as Si0 2, A 120 3, Ti0 2, Zr0 , Fe20 3, in particular metal oxides, and semi¬
conductors such as ZnS, CdS, CdSe to name some. An example for such filled polymer is a
cyclic olefin polymer filled with Ti0 2.
The objects of the present invention are also solved by a method of providing a polymeric
substrate with a glass-like surface, in particular an etched-glass-like surface, said method
comprising the following steps:
a) providing a polymeric substrate which, preferably, is made of a polymeric substrate mate¬
rial according to the present invention;
and one of the following steps:
bl) coating a surface of said polymeric substrate with SiOx; x being in the range of from 1 to
2;
b2) coating a surface of said polymeric substrate with a SiOx-precursor and converting said
SiCvprecursor into SiOx, x being in the range of from 1 to 2;
b3) endowing a surface of said polymeric substrate with a glass-like surface morphology, in
particular an etched glass-like surface morphology, by
coating a surface of said polymeric substrate with a coating material having increased
roughness in comparison to the uncoated surface of said polymeric substrate and/or in¬
creased hydrophilicity, said coating material preferably being a polymer, and/or
by plasma-treating and/or reactive ion etching (RIE) and/or by treatment with UV
Ozone cleaner of a surface of said polymeric substrate or said coated polymeric sub¬
strate, preferably using argon, oxygen, H20 , H2, fluorinated methane gases, such as
CF4, CHF3, CH2F2, or a mixture of or a sequential use of any of the foregoing, to in¬
duce roughness and hydrophilicity on said surface;
b4) a combination of bl), b2) and b3) in any order.
In one embodiment, plasma treatment occurs with a frequency in the range of from 0.1 KHz
to 100 GHz. In one embodiment, an etched-glass like surface morphology can also be
achieved by a sequential use of plasma-treatment and reactive ion etching. In one embodi¬
ment, the plasma treatment occurs with a frequency in the range of from 1 KHz to 999 KHz.
In another embodiment, plasma treatment occurs with a frequency in the range of from 1
MHz to 999 MHz. In yet another embodiment, plasma treatment occurs with a frequency in
the range of from 1 GHz to 100 GHz. In one embodiment, the plasma treatment may involve
a single step or multiple steps of plasma treatment. The term "Ar/02 -plasma", as used herein,
in one embodiment, refers to a plasma treatment using argon, followed by plasma treatment
using oxygen, or vice versa. In another embodiment, it may refer to a plasma treatment using
a mixture of argon and oxygen in a single step. In yet another embodiment, the term refers to
multiple steps of plasma treatment using argon, followed by multiple steps of plasma treat¬
ment using oxygen, or vice versa. In yet another embodiment, the term refers to a single step
or multiple steps of plasma treatment using argon, followed by a single step or multiple steps
of plasma treatment using oxygen, or vice versa.
In one embodiment, the plasma treatment is an Ar/0 2-plasma treatment.
In one embodiment, the plasma treatment is an Ar/UV Ozone treatment.
The term Ar/UV Ozone treatment as used herein, in one embodiment refers to a plasma
treatment using Argon, followed by UV Ozone treatment, or vice versa. In another embodi¬
ment, it may refer to a plasma treatment using Argon, together with UV Ozone treatment in a
single step. In yet another embodiment, the term refers to multiple steps of plasma treatment
using Argon, followed by multiple steps of UV Ozone treatment, or vice versa. In yet another
embodiment, the term refers to a single step or multiple steps of plasma treatment using Ar¬
gon, followed by a single step or multiple steps of UV Ozone treatment, or vice versa.
In one embodiment, step bl), b2) and/or b3) is performed
by physical vapor deposition methods, such as:
- thermal deposition (physical vapor deposition)
- electron-beam (E-gun) deposition
- sputtering,
- by chemical vapor deposition (CVD),
- by film growth on the surface, such as electroless plating or electrochemical deposition,
- spray coating
- dip coating
- gas-phase deposition
- roll-to-roll deposition,
- screen printing,
- doctor blading,
- wet coating,
- dynamic coating,
- CVD,
or a combination of several of the foregoing.
In one embodiment, the converting in step b2) is performed by one of:
- annealing, preferably thermal annealing
- irradiating with electromagnetic radiation, such as infrared or UV, preferably UV
- treatment in solution with water or base or acid, or a combination of water and base, or a
combination of water and acid.
In one embodiment, said SiO -precursor is selected from the group comprising
i) alkoxy- or alkyl-chlorosilane, S1X4, trisiloxane compound Si30 2X6, X being, independently,
at each occurrence OR or halogen, R being alkyl, alkyl being preferably C -C20-alkyl, pref¬
erably ethyl or methyl;
ii) polysilazane, such as perhydropolysilazane, -[Si(H) 2-N(H)-] n, n being selected from 3 to
10000, or polyorganosilazane -[Si(R) 2-N(R)-] n, R being alkyl, alkyl being preferably
alkyl, n being selected from 3 to 100 000;
iii)a sol-gel containing SiO particles, said particles preferably having a diameter of about 1
nm to 10 mhi, preferably 10 nm to 100 nm, suspended in a solvent-based matrix , the solvent
being preferably an alcohol, preferably ethanol.
In one embodiment, in steps bl) - b3), together with said SiOx, SiOx-precursor or said poly¬
mer, additional materials are coated on said surface of said polymeric substrate, said addi¬
tional material being selected from Si2N3, A 120 3, B20 3, Ti0 2, Na20 , CaO, K20 , S0 3, MgO,
Fe20 3, SiOx (x being < 2).
In one embodiment, said coating material, in particular said polymer, having increased rough¬
ness and/or intrinsic porosity is selected from polytetrafluoroethylenes (Teflon), such as 2,2-
bistrifluoromethyl-4,5-difluoro-l,3-dioxoletetrafluoroethylene copolymer (designated Teflon
AF 2400 by Du Pont), nafion, polysulfones, poly(oxy-2,6-dimethyl-l,4-phenylene), and the
group of substituted acetylene polymers, like poly[l-(trimethylsilyl)-l-propyne] (PTMSP),
poly [1-(trimethylgermyl)- 1-propyne] , poly(4-methyl-2-pentyne), poly [1-phenyl-2-(ptrimethylsilylphenyl)
acetylene], and poly[2,4,5-tris(trifluoromethyl)-phenylacetylene].
The term "nafion", as used herein, is meant to refer to a sulfonated tetrafluoroethylene based
fluoropolymer-copolymer. In nafion, perfluorovinylether groups which are terminated with
sulfonate groups, are incorporated into a tetrafluoroethylene (Teflon) backbone. The sulfonic
acid functional groups self-organize into hydrophilic water channels of a few nanometres in
diameter
In one embodiment, said method further comprises a step
c) post-treating said substrate by one or several of
- drying,
- water treatment,
- tempering,
- sintering,
- solvent treatment,
- plasma treatment,
- reactive ion etching,
-treatment with UV-Ozone cleaner,
and any combination of the foregoing.
The term "treatment with UV-Ozone cleaner" or "UV Ozone treatment", as used herein, is
meant to refer to a treatment involving the irradiation by short wavelength UV radiation, typi¬
cally in the range of from 180 nm to 260 nm, usually also using two ore more different wave¬
lengths from within such range. Such irradiation leads to in-situ generation of atomic oxygen
and ozone which in term react with contaminant substrates. Hence UVO cleaner treatment
will result in the cleaning of a surface.
The method according to the present invention may also involve one or several "pretreatment"
steps, which is (are) performed on the substrate before any coating step is performed.
Such pretreatment may be a plasma treatment, a heat treatment, an exposure to chemicals,
and activation of the substrate surface by endowing it with chemical functional groups etc.
Such pretreatment step(s) may facilitate the subsequent steps performed thereafter and may
improve the quality of the substrate.
In one embodiment, said method is performed at least on a first polymeric substrate made of a
material, as defined further above, wherein said first polymeric substrate thereby is provided
with at least a first glass-like surface, in particular a first etched-glass-like surface, said meth¬
od further comprising the bonding step:
bonding said first substrate to a second substrate by bringing said first glass-like surface, in
particular etched-glass-like surface in contact with a surface of a second substrate, by pressing
said first and second substrate together, preferably by a pressure in the range of from 0.2
N/mm 2 to 5 N/mm 2, more preferably in the range of from 0.5 N/mm 2 to lN/mm 2, for a time in
the range of from 0 s to 600 s, more preferably in the range of from 30 s to 120 s and by ex¬
posing said first and second substrate to a temperature in the range of from 40°C to 200°C,
preferably 60°C to 120°C, and/or exposing said first and second substrate to a vapor of sol¬
vent immediately before pressing to increase their bonding to each other.
In one embodiment, said first polymeric substrate is a solid substrate, and wherein said second
substrate is either a solid substrate or a flexible foil.
In one embodiment, said second substrate is a polymeric substrate made of a material as de¬
fined further above, or said second substrate is made of a material selected from glass, quartz,
silicon nitride and silicon oxide, or, if said second substrate is a flexible foil, said second sub¬
strate is made of polyolefines, polyethers, polyesters, polyamides, polyimides, polyvinylchlorides,
polyacrylates; including their modifications, derivates, derivatives and copolymers;
more specifically one of the list containing acrylntiril-butadien-styrole (ABS), cyclo-olefinpolymers
and copolymers (COC/COP), Polymethylene-methacrylate (PMMA), Polycarbonate
(PC), Polystyrole (PS), Polypropylene (PP), Polyvinylchloride (PVC), Polyamide (PA), Poly¬
ethylene (PE), Polyethylene-terephtalate (PET), Polytetrafluor-ethylene (PTFE), Polyoxymethylene
(POM), Thermoplastic elastomers (TPE), thermoplastic polyurethane (TPU),
Polyimide (PI), Polyether-ether-ketone (PEEK), Polylactic acid (PLA), polymethylpentene
(PMP), and derivatives thereof, wherein said polymer is optionally filled with an inorganic
material such as carbon black, oxides, such as Si0 2, A 120 , Ti0 2, Zr0 2, Fe20 3, in particular
metal oxides, and semiconductors, such as ZnS, SdS, SdSe.
In one embodiment, one or both of said first substrate and said second substrate has a channel
or groove or recess or hole in it, which, preferably, forms a conduit at the interface between
said first and second glass-like surfaces, wherein more preferably, said conduit extends from
one edge to another edge of said first or second substrate or from one hole of one of the sub¬
strates to another hole of one of said substrates, thus allowing the flow-through of a liquid
through said conduit.
In one embodiment, the method according to the present invention is performed on said first
polymeric substrate and on at least a second polymeric substrate made of a material according
to the present invention and, optionally, on a third and/or further polymeric substrates, each
being made of a material according to the present invention, wherein said first and second
polymeric substrate and, if present, said third and further polymeric substrates are subse¬
quently bonded to each other by the bonding step as defined further above.
In one embodiment, one, two or more of said polymeric substrates have a channel or groove
or recess or hole, which forms a conduit at the interface between said substrates, wherein,
preferably, said conduit extends from one edge to another edge of said substrate(s) or from
one hole of one of the substrates to another hole of one of said substrates, thus allowing the
flow-through of a liquid through said conduit.
The objects of the present invention are also solved by a method of manufacturing a chip,
preferably, a microfluidic chip, said method comprising:
a) providing at least a first polymeric substrate, made of a material as defined further above,
and a second substrate, said second substrate being either a solid substrate or a flexible foil
and being made of a polymeric material as defined further above or of a material selected
from glass, quartz, silicon nitride, and silicon oxide, or, if said second substrate is a flexible
foil, being made of: polyolefines, polyethers, polyesters, polyamides, polyimides, polyvinylchlorides,
polyacrylates; including their modifications, derivates, derivatives and copolymers;
more specifically one of the list containing acrylnitril-butadien-styrole (ABS), cyclo-olefinpolymers
and copolymers (COC/COP), Polymethylene-methacrylate (PMMA), Polycarbonate
(PC), Polystyrole (PS), Polypropylene (PP), Polyvinylchloride (PVC), Polyamide (PA), Poly¬
ethylene (PE), Polyethylene-terephthalate (PET), Polytetrafluor-ethylene (PTFE), Polyoxymethylene
(POM), Thermoplastic elastomers (TPE), thermoplastic polyurethane (TPU),
Polyimide (PI), Polyether-ether-ketone (PEEK), Polylactic acid (PLA), polymethylpentene
(PMP), wherein at least one of said first and said second substrate have a channel or recess or
groove or hole in it; and either
bl) performing the method according to the present invention on at least said first substrate,
preferably on both said first and second substrate, and subsequently performing the bonding
step as defined further above thereon; or
b2) bringing said first and said second substrate in contact with each other and bonding them
with each other by the bonding step as defined further above to form an assembly of said first
and second substrate, and subsequently, performing the method of providing a polymeric sub¬
strate with a glass-like surface in accordance with the present invention on said assembly, in
particular on the conduit(s) which is (are) formed inside said assembly.
In one embodiment, step a) includes providing a plurality of substrates, according to the pre¬
sent invention, and steps bl) or bl) are performed on said plurality of substrates, thus result¬
ing in a chip comprising a plurality of substrates which are stacked on one another.
The objects of the present invention are also solved by a polymeric substrate produced by the
method of providing a polymeric substrate with a glass-like surface according to the present
invention.
The objects of the present invention are also solved by a chip, in particular a microfluidic
chip, made of two substrates, at least one of which, preferably at least two of which are poly¬
meric substrates in accordance with the present invention as defined further above, said chip
having at least one conduit going through said chip, said conduit preferably having a smallest
dimension being in the range < 500 mp , preferably < 200 mh .
In one embodiment, the chip according to the present invention is made of one polymeric sub¬
strate as defined further above according to the present invention and either
- a) another such polymeric substrate as defined further above according to the present inven¬
tion,
- or b) another substrate which is a flexible foil as defined further above,
- or c) another substrate which is made of a material selected from glass, quartz, silicon ni¬
tride, and silicon oxide,
- or d) a plurality of substrates, wherein said plurality of substrates is composed of a plurality
of substrates of the type defined in a) or b) or c), or is composed of a plurality of substrates of
any combination of the types defined in a) - c).
In one embodiment, said at least one conduit is filled with a matrix suitable for the analysis
and/or detection and/or separation and/or transport of analytes, preferably of biological cells,
or biological macromolecules and/or their respective mono/oligomers, such as nucleic acids,
nucleotides, proteins, peptides, amino acids, carbohydrates, polysaccharides, oligosaccha¬
rides, monosaccharides, lipids, fats, and fatty acids.
In one embodiment, said matrix is a gas phase, a liquid, a dispersion of solid particles in a
liquid, or a gel, preferably a polyacrylamide gel or an agarose gel or a starch gel or a sucrose
gel.
The objects of the present invention are also solved by a chip, in particular a microfluidic
chip, produced by the method according to the present invention.
The objects of the present invention are also solved by a kit for manufacturing a chip, in par¬
ticular a microfluidic chip, by the method according to the present invention, said kit compris¬
ing at least a first substrate and a second substrate,
wherein at least one of said first and second substrate has a channel or recess or groove or
hole in it, said kit further comprising one or several agents to perform the method according
to the present invention on said substrates.
In one embodiment, said first and second substrate is made of a material selected from poly¬
mers of synthetic or natural origin, preferably injection-moldable polymers, more preferably
one of the material classes containing: polyolefines, polyethers, polyesters, polyamides, polyimides,
polyvinylchlorides, polyacrylates; including their modifications, derivates, derivatives
and copolymers; more specifically one of the list containing acrylntiril-butadien-styrole
(ABS), cyclo-olefin-polymers and copolymers (COC/COP), Polymethylene-methacrylate
(PMMA), Polycarbonate (PC), Polystyrole (PS), Polypropylene (PP), Polyvinylchloride
(PVC), Polyamide (PA), Polyethylene (PE), Polyethylene-terephtalate (PET), Polytetrafluorethylene
(PTFE), Polyoxymethylene (POM), Thermoplastic elastomers (TPE), thermoplastic
polyurethane (TPU), Polyimide (PI), Polyether-ether-ketone (PEEK), Polylactic acid (PLA),
and polymethylpentene (PMP) , and derivatives thereof, wherein said polymer is optionally
filled with an inorganic material such as carbon black, oxides, such as Si0 2, A 120 3, Ti0 2,
Zr0 2, Fe20 3, in particular metal oxides, and semiconductors, such as ZnS, SdS, SdSe.
The objects of the present invention are also solved by a kit for the analysis and/or detection
and/or separation and/or transport of analytes, preferably of biological macromolecules and
their respective mono/oligomers, said kit comprising:
the chip according to the present invention, and, optionally, one or several agents, such as e.g.
buffers, necessary for the analysis and/or detection and/or separation and/or transport of ana¬
lytes.
The objects of the present invention are also solved by the use of a polymeric substrate or of a
chip, in particular a microfluidic chip, according to the present invention in a method of ana¬
lyzing, detecting, separating and/or transporting analytes, preferably biological cells, or bio¬
logical macromolecules and/or their respective mono/oligomers, in particular in a method of
electrophoresis, a method of sequencing, an assay method for the detection of an analyte, or a
method of flow cytometry.
The term "assay", as used herein, is meant to refer to a procedure for testing or measuring the
activity or presence of a drug or biochemical or molecule or substance in an organism or or¬
ganic sample. A quantitative assay may also measure the amount of a substance in a sample.
A qualitative assay may also detect the presence of a drug or biochemical or molecule or sub¬
stance or organism. Bioassays and immunoassays are among the many varieties of specialized
biochemical assays. Other assays measure processes such as enzyme activity, antigen capture,
stem cell activity, gene expression, genotyping, DNA/RNA analysis and competitive protein
binding, all of which assays are meant to be encompassed by the term "assay".
The objects of the present invention are also solved by a method for analysing, detecting, sep¬
arating and/or transporting analytes, preferably biological macromolecules and/or their re¬
spective mono/oligomers, comprising:
- providing, in any order, a chip or polymeric substrate according to the present invention, and
a sample containing analyte(s) to be analysed, separated and/or transported, said chip includ¬
ing a matrix as defined further above, said polymeric substrate having a matrix as defined
further above attached,
- applying said sample to said matrix,
- applying a voltage to said matrix, said voltage being sufficient to cause the migration or dif¬
fusion or flow of the analyte(s) through said matrix,
- irradiating or ablating or desorbing or ionising the analyte(s), and, optionally, further
- analysing the analyte(s).
The objects of the present invention are also solved by the use of a substrate according to the
present invention for manufacturing a chip, in particular a microfluidic chip, for the analysis
and/or separation and/or transport of analytes.
The term "etched-glass-like", as used herein, is meant to refer to a surface which mimics the
surface of etched-glass, in one of the aforementioned characteristics, without, however, neces¬
sarily having been etched before. The process of etching glass is well known to a person
skilled in the field, as is the resultant glass-product of such process, which is known to be
characterized by an increased surface roughness and other characteristics, mentioned above,
in comparison to untreated, i.e. non-etched glass. The term "thin film" as used herein, refers
to a film or layer having a thickness in the range from 0.1 nm to 1 m h, more preferably 1 nm
to 100 nm.
The term "glass-like", as used herein, is meant to refer to a surface which mimics the surface
of glass, in one of the aforementioned characteristics, without, however, being a glass-surface.
In one embodiment, such glass-like surface is formed by a polymer.
The term "SiOx", or „silicon oxide" as used herein, is meant to refer to any form of silicon
oxide, including, but not limited to all crystalline forms, polycrystalline forms, and/or amor¬
phous forms, of silicon oxide, with varying amounts of oxygen present, and also including
incompletely converted forms of SiOx-precursor which may include organic groups. The term
"SiOx" also includes Si0 2 and SiO, without being limited thereto. In one embodiment, the
variable "x" is in the range of from 1 to 2 and also includes non-integer values.
The term "increased", as used herein in combination with "porosity", "roughness" or another
quality is meant to refer to such quality being larger in comparison to an untreated substrate.
In one embodiment, said SiOx-precursor is dissolved in a solvent or is present in sol-gel form
when being coated in step b2).
In one embodiment, the method according to the present invention is performed on a first pol¬
ymeric substrate and a second polymeric substrate or on a plurality of polymeric substrates,
and wherein said first substrate and said second substrate thereby are provided with a first and
second glass-like or etched-glass-like surface, respectively, said method further comprising
the step:
bringing said first and said second glass-like or etched-glass-like surface in contact with each
other by pressing said first and second substrate together, preferably by a pressure in the range
from 0.2 to 5 N/mm 2, more preferably in the range from 0.5 to 1 N/mm 2, for a time in the
range from 10 to 600 s, more preferably in the range from 30 to 120 s, and by exposing said
first and second substrate to a temperature in the range of from 40°C to 200°C, preferably
60°C to 80°C and/ or exposing said substrates to a vapour of a solvent immediately before
pressing to increase their bonding to each other. It should be noted that the above mentioned
first and second glass-like or etched-glass-like surfaces may also be part of a plurality of pol¬
ymeric substrates and there are further glass-like or etched-glass like surfaces obtained there¬
by. These may also be subsequently brought in contact with one another and thus bonded to
one another. This may for example result in a multiple stacks chip. The present invention also
encompasses a structure where the method according to the invention is performed on one
first polymeric substrate, and on a second polymeric laminar foil. These may also be subse¬
quently be brought in contact with one another and thus bonded to one another. This may for
example result in a laminar type chip. The present invention also encompasses a structure
where the method according to the invention is performed on one polymeric substrate, and the
surface obtained thereby is subsequently bonded to a surface of a e.g. glass-substrate or a
quartz substrate etc. In this manner the production of hybrid structures, i.e. a glass substrate
bonded to a polymeric substrate, becomes also possible.
It should be noted that one of said first and second substrates is a polymeric substrate as de¬
fined further above, and, in one embodiment, the other substrate is also a polymeric substrate.
In another embodiment, the other substrate is a polymeric foil; in yet a further embodiment,
the other substrate is a glass substrate or a quartz substrate, as an example.
The term "kit", as used herein, is meant to refer to an assembly of parts allowing the manufac¬
ture or use of a chip as defined above. In its simplest form, this kit comprises one or several
agents to perform the method of providing a polymeric substrate with a glass-like surface, in
particular an etched-glass-like surface according to the present invention, or to perform an
analysis, detection separation and/or transport of analytes. In one embodiment, that kit may
also additionally comprise at least one polymeric substrate, as defined further above. In one
embodiment, said kit comprises, in addition to said at least one polymeric substrate, a further
polymeric substrate as defined above, or a polymeric foil or a glass substrate.
The objects of the present invention are also solved by the use of a substrate as defined above
for manufacturing a chip, in particular a microfluidic chip.
The term "chip", as used herein, is meant to refer to a device comprising at least one substrate
which allows for the accommodation and/or the transport of a sample, preferably a liquid
sample. Such accommodation or transport of a sample may for example be achieved in that
such substrate comprises a well and/or channel and/or groove and/or recess or it may com¬
prise a conduit. In one embodiment, the chip comprises at least one well. Such well may, for
example, accommodate a liquid sample, or it may accommodate a solid sample, such as parti¬
cles, in particular microparticles, which may be covered with a ligand, e. g. an antigen.
The term "microfluidic chip", as used herein, typically refers to such chip in the aforemen¬
tioned sense, wherein, however, the dimensions of at least some of the wells, channels,
grooves, recesses or conduits etc. are on the micrometer range.
In one embodiment, the term "glass-like", as used herein in the context of a substrate, is
meant to refer to a scenario wherein such substrate, in particular the surface of such substrate,
mimics one or several properties of glass, in particular the chemical content (Si, O) and com¬
position, homogeneity, roughness (<3 nm RMS), porosity, hydrophilicity (water contact angle
<50° for cleaned glass), surface energy and adsorption affinity, surface functionality, chemi¬
cal and physical surface reactivity, and surface charge (Zeta potential < 0 V for pH > 2, pref¬
erably for pH > 3), ion-exchange capability, proton conduction in solution due to formation of
water channels inside the substrate, high inner surface, preferably > 100 m2g 1, more prefera¬
bly > 500 m g , high permeation of gases, without being limited thereto and without neces¬
sarily having been etched itself.
The term "an etched glass-like", as used herein in the context of a substrate, is meant to refer
to a scenario wherein such substrate, in particular the surface of such substrate, mimics one or
several properties of etched glass, in particular the chemical content (Si, O) and composition,
homogeneity, roughness (>3 nm RMS), porosity, hydrophilicity (water contact angle <50° for
cleaned glass), surface energy and adsorption affinity, surface functionality, chemical and
physical surface reactivity, and surface charge (Zeta potential < 0 V for pH > 2, preferably for
pH > 3), ion-exchange capability, proton conduction in solution due to formation of water
channels inside the substrate, high inner surface, preferably > 100 m g_ 1, more preferably >
500 m2g , high permeation of gases,without being limited thereto, and without necessarily
having been etched itself.
The term "biological macromolecule", as used herein, is meant to refer to macromolecules
that typically occur in living matter and organisms, for example nucleic acids, proteins, pep¬
tides, carbohydrates, polysaccharides, lipids, and fats. Their respective mono/oligomers are
the corresponding nucleotides, peptides, amino acids, sugars, fatty acids in their respective
mono/oligomeric forms. Also encompassed by the term "biological macromolecule", as used
herein, are macromolecular assemblies, such as viruses, cell organelles, ribosomes, mitochon¬
dria, chromosomes and other comparable structures. The substrates and chips according to the
present invention find use in the analysis, detection, separation, and/or transport of any of the
foregoing.
The present inventors have surprisingly found that it is possible to combine the positive quali¬
ties of polymeric substrates with the positive characteristics of glass surfaces, in particular
etched-glass-surfaces by endowing such polymeric substrates with a glass-like surface, in
particular an etched-glass-like surface. Preferably, the polymeric substrate is a carbonpolymer-
based substrate. In one embodiment, the substrate is not polydimethylsiloxane.
In a preferred embodiment, the polymeric substrates in accordance with the present invention
have a zeta potential which is negative, i.e. < 0V for pH > 2, more preferably > 3. If one
measures the zeta potential in dependency on the pH, the measured values for the polymeric
substrates in accordance with the present invention are comparable to the respective potentials
measured with glass or etched glass. In some embodiments, the zeta potential of the substrates
in accordance with the present invention is more negative than a glass surface.
Furthermore, the polymeric substrates in accordance with the present invention show the
same behaviour as glass, when in contact with SDS, as measured, for example by the respec¬
tive zeta potential. In one embodiment, the zeta potential of the polymeric substrate in accor¬
dance with the present invention is not substantially affected by the presence of SDS. This is
in contrast to polymeric substrates not according to the present invention, where the zeta po¬
tential can be altered drastically by the presence of SDS. Preferably, the substrates in accor¬
dance with the present invention have a zeta potential < 0 at a pH of 3 or more, and the pres¬
ence of SDS shifts the zeta potential to no more than twice as negative values as the zeta po¬
tential in the absence of SDS.
In one embodiment, the surface of the polymeric substrate is coated with Si0 2 by means of
thermal deposition, heated electrically or by an electron-gun, or by sputtering. In another em¬
bodiment, a Si0 2-precursor, such as TEOS, OTCS or TTBS-OH or perhydropolysilazane is
applied to the surface of the polymeric substrate and subsequently converted into Si0 2 by ap¬
propriate post treatments, such as annealing, irradiating with energetic electromagnetic radia¬
tion, such as UV or treatment with water or base or acid or combinations thereof. In doing so,
the polymeric substrate is endowed with a surface that has a characteristic, such as chemical
composition that mimics the surface of glass or etched glass. Such substrate is amenable to
bonding with other substrates or laminar foils, e. g. such substrates which have been treated in
the same manner. Such substrate endowed with a surface that has a characteristic mimicking
the surface of glass or etched glass is, however, also amenable to bonding with other sub¬
strates not treated according to the present invention.
The term "polymer or coating material having intrinsic porosity", as used herein, is meant to
refer to a polymer or coating material which by its nature is porous and has a high inner sur¬
face. Preferably, the dimensions of the pores are in the range of 0.5 nm to 50 nm, preferably
in the range of 1 nm to 10 nm and the inner surface is larger than 100 m g . If a polymeric
substrate is provided with a surface of such a polymer having intrinsic porosity, the morphol¬
ogy, in particular the porosity of a glass or an etched glass-surface, is mimicked. The term
"polymer or coating material having increased roughness", as used herein, is meant to refer to
a polymer which by its nature has a relatively high RMS surface roughness after its deposition
as a thin film. Preferably, the RMS surface roughness is in the range of 0.1 nm to 1mhi, pref¬
erably in the range of 1 nm to 100 nm, as measured by AFM with standard AFM tips. The
term "increased roughness", as used in this context is meant to refer to a roughness that is
increased in comparison to the uncoated or not treated surface of a substrate.
The various embodiments of providing a glass-like or an etched glass-like surface may also be
combined in a sense that both the chemical composition as well as the morphology and
roughness of glass or etched glass is mimicked thereby.
The substrates in accordance with the present invention have a long time stability, as for ex¬
ample measured by their respective contact angle. For example, in one embodiment, the con¬
tact angle of a substrate surface treated in accordance with the present invention stays below
50 degrees for a period of up to 60 days and more. This makes the substrates in accordance
with the present invention amenable to a use in industrial processes and repeated uses.
The inventors have also found that, after two substrates have been treated in the afore¬
mentioned manner in accordance with the present invention, they may be bonded together,
thus forming an assembly of two substrates having an interface. If at least one of these sub¬
strates encompasses a channel or groove or recess and is contacted with another substrate or
foil, such channel or groove or recess will form a conduit in the assembly of the two sub¬
strates. By choosing appropriate extensions of the channel in one of the substrates, for exampie
by having the channel extend from one end of the substrate to another end, an appropriate
conduit can be formed which allows the flow-through of liquid through the assembly. In this
manner, fluidic chips can be produced, in particular microfiuidic chips. The term "microfluidic
chip", or "microchip" as used herein, is meant to refer to a chip which has one or several
conduits extending there through, which conduits allow the flow-through of liquid or gel or a
polymer dye matrix. Preferably, such conduits preferably have a width in the range of 10 mp
- 200 m , and a depth in the range of 1 mp - 100 m . Additionally, there may also be larger
structures on/in that chip, such as reservoirs for liquids etc. The term "conduit", as used here¬
in, is meant to refer to a hollow structure at an interface between two substrates through
which a liquid can flow. A conduit is formed by a channel or recess or groove in at least one
of the substrates. Thus, the term "channel", "groove", or "recess" refers to a hollow structure
on the surface of a single substrate. If such surface is then subsequently contacted with an¬
other substrate or foil and is effectively thereby covered, a conduit is formed. Sometimes, a
substrate having a channel or recess or groove in it is herein also referred to as a channel
plate. The substrate that covers such channel plate is also herein sometimes referred to as a
cover plate or well plate (if it has holes on the surface, not the edge, as an entrance for the
analyte). In accordance with embodiments of the present invention, a chip or microfiuidic
chip may thus for example be formed by two substrates being bonded together or laminated,
wherein at the interface, a conduit is formed by one or several channels on at least one of the
substrates. Such substrates together are also herein sometimes referred to as an "assembly of
substrates". In such assembly of substrates, the two substrates are interfacing each other with
their respective glass-like or etched glass-like surfaces.
The order of the manufacturing steps for microchips may be either, first, surface treatment of
one or both substrates and/or foil, and subsequent bonding, or the other way around, or a
combination of the two, i.e., some parts of the treatment before bonding, others after.
Polymeric substrate materials are commercially available from various manufacturers. Exam¬
ples of suitable materials are:
Trogamid CX7323®, PMMG CMG302®, Delpet 70NH, Zeonor 1060R®, Zeonex® 480, PC
black Makrolon® 2405, Arton® D4540, Zeonor® 1060R + 2% black, PP Dow H734-52®,
TPX®, PC®, PP®, Topas® 8007X1 1, PC-Teijin®, PC-MEP®, PC-Dow®.
In accordance with the present invention, glass-like or etched-glass-like properties on poly¬
meric substrates are achieved by appropriate coatings or surface modifications. This allows
the manufacture and use of microchips made of such polymeric substrates without further
needs to change the experimental protocols for their respective applications as compared to
glass microchips, i.e. they can be used as direct substitution. Moreover, it is possible to finetune
the coating properties by controlling the interface and the interaction of biomolecules
at/with the interface of the polymer substrate.
The coatings and surface modifications in accordance with the present invention are perma¬
nent and stable over time and they are easily integrated in production processes, since they are
not labor-intense. In one embodiment, the thicknesses of the layers of coatings can be varied.
In one embodiment, the thickness of a coating layer is homogenous and is in the range of
from 0,1 nm to 500 nm, preferably 5 nm to 200 nm. Such manufacture of surface layers hav¬
ing a thickness in the afore-mentioned ranges is of crucial importance in microfluidic applica¬
tions, since their respective conduits for flow-through of liquid or gel are in the micrometer
range. Consequently, the surface-modifying layers must be much thinner than the channel
height.
The method in accordance with the present invention allows the easy production of disposable
substrates and chips. Moreover, the substrates and chips produced in accordance with the pre¬
sent invention are compatible with existing experimental protocols for glass chips and meth¬
ods for mass-production.
In the following, reference is made to the figures, wherein
figure 1 shows transmission curves of (a) Si0 2-layers of different thickness evaporated
on Zeonor 1060R and (b) Zeonor 1060R and glass as reference. The transpar¬
ency is not strongly dependant on layer thickness (5 nm to 100 nm),
figure 2 shows results of durability tests of Si0 2 layer on Zeonor 1060R deposited with
different methods. The contact angle is more stable on low value in the case of
E-gun deposition.
figure 3 shows an example image of a substrate having appropriate channels therein,
with channel dimensions before and after coating with Si0 2.
figure 4 shows a scheme for applying silane and siloxane as a liquid-Si0 2 precursor,
preceded by plasma treatment, such as oxygen, or H20 , or others,
figure 5 shows examples of liquid Si0 2 precursors, a) Tetraethyl orthosilicate (TEOS)
b) Octachlorotrisiloxane (OTCS), and c) Hydroxymethyltriethoxysilane
(TTBS-OH)
figure 6 shows an AFM image (topography) of a polymeric substrate coated with TEOS
in accordance with the present invention; the surface roughness is < 0.5 nm, i l
lustrating a homogeneous coating,
figure 7 shows the results of XPS to confirm the presence of Si0 2 which has been silanized
with TEOS on the substrate surface,
figure 8 shows a bonding scheme of a well plate b and a channel plate c, both plates
having been coated a in accordance with the present invention and comprising
a glass-like and /or an etched-glass-like surface. The result is a bonded chip d.
figure 9 shows an optical microscope image of a cross section of a bonded conduit
formed by two substrates that have been treated in accordance with the present
invention and have been provided with a glass-like, in particular an etchedglass-
like surface; there is no deformation of the structure that can be seen in
the optical microscope image, hence, the dimensions of the channel/conduit do
not change upon coating and bonding.
figure 10 shows an optical microscope image of conduits formed via bonding of two
substrates comprising surfaces treated in accordance with the present invention.
The channels are filled with a liquid of dark color. No leakages of the liquid
can be discerned,
figure 1 shows the structure of a liquid precursor, perhydropolysilazane,
figure 12 shows an AFM-image of a substrate coated with perhydropolysilazane after
NH4OH vapor treatment, indicating a homogeneous coating,
figure 13 shows an optical microscope image of conduits coated with perhydropolysi¬
lazane and treated with NH4OH vapor. The coating does not change the dimen¬
sions of the conduits on a mpi scale,
figure 14 shows a FTIR absorption spectrum of a COC substrate coated with poly[l-
(trimehylsilyl)-l-propyne] (PTMSP), the COC background spectrum has been
subtracted; the presence of the coating is identified by the characteristic pres¬
ence of absorption of chemical groups of PTMSP,
figure 15 shows Kelvin probe force microscopy scans of (a) PTMSP film, (b) glass, (c)
COC, and (d) COC coated with polysilazane, charged by 1 s voltage pulses ap¬
plied to standard conducting AFM probe in contact to the samples. The strong
charging (white spots) in (a) and (d) indicates, that water ions can penetrate in¬
to the volume of the films, similar to the charging observed on the glass film
(b). The uncoated COC film (c) is not charged strongly,
figure 16 shows AFM topography images of a untreated PMMA substrate (a), and after
Ar/0 2 plasma treatment (b); the surface roughness increases significantly upon
plasma treatment (from 4 nm to 25 nm RMS). The receding water contact an¬
gle on (b) is much lower (<10°) than the advancing contact angle (50°), indicat¬
ing strong roughness,
figure 17 shows an electrophoretic separation of DNA 7500 analyte obtained with
a PMMA chip in accordance with the present invention (a) and the results of an
electrophoretic separation using a conventional glass chip for comparison (b),
figure 18 shows an electrophoretic separation of Bovine Serum Albumine analyte ob¬
tained with COC chips which is wet-coated with Si0 2 sol-gel in accordance to
the present invention (a) and an electrophoretic separation using a conventional
glass chip for comparison (b).
figure 19 shows an optical microscope image of conduits treated with Ar/02 plasma. The
treatment allows a successful bonding.
figure 20 shows an optical microscope image of conduits treated with Ar plasma / UVOzone
cleaner treatment. The treatment allow a successful bonding.
figure 21 shows an (a) optical microscope image of a cross section of a COP bonded
conduit formed by two substrates that have been treated with Ar plasma AJVOzone
cleaner, there is no deformation of the structure that can be seen in the
optical microscope image, hence, the dimensions of the channel/conduit do not
change upon treatment and bonding . Figure 1 (b) shows an electrophoretic
separation of DNA 7500 analyte obtained with a COP chip treated with Ar
plasma / UV-Ozone cleaner.
figure 22 shows AFM topography scans of (a) Ar plasma /UV-Ozone cleaner treatment
on PMMA and (b) on COP; the surface roughness increases significantly upon
plasma treatment to (a) 16 nm rms roughness, and (b), to 7 nm rms roughness.
Figure 23 shows an SEM image of a COP assay substrate
figure 24 shows contact angle measured in difference fields of an assay substrate that
have been treated in accordance with the present invention, repeated in differ¬
ent days after treatment, in particular a Ti0 2 filled COP substrate treated with
Ar/02 plasma show after 114 days a contact angle below 65°. The untreated
Ti0 2 filled COP substrate have a contact angle of 110-120°.
figure 25 shows (a) three 80 mm profilometer scan lines along the x-direction, offset in
the y-direction by 5 mm, on a COP slide after treatment. The undulations in
height are all below 1 m i on 1 mm range, and show (b) the contact angle
measured in difference fields of an assay substrate that have been treated in ac¬
cordance with the present invention, repeated in different days after treatment,
in particular a COP substrate coated with Si02 thin film show after 115 days a
contact angle below 30°.
figure 26 shows AFM topography scans of (a) molded wells in a plastic substrate, and
(b), the same plastic substrate after evaporation of 20 nm Si0 2. Morphology
and roughness of the wells are not affected by the Si0 2 evaporation,
figure 27 shows AFM topography scans of 1 x 1 mh of a COC substrates after Ar/02
plasma treatment, the roughness is increasing from 0.8 nm rms of the bare sub¬
strate to 6 nm rms after the treatment.
figure 28 shows an XPS spectrum of (a) an untreated and (b) an Ar/02 plasma treated
COC flow cytometry chip. The data confirm that the treatment did not change
the chemical composition of the polymeric substrate,
figure 29 shows AFM topography scans of (a) 10 x 10 m , and (b), of 1 m area of
COC substrates before and after TEOS coating.
figure 30 shows an XPS spectrum of (a) an untreated and a (b) TEOS coated COC flow
cytometry chip. The data confirm the presence of Si0 2 on the substrate surface
of the chip coated with TEOS,
figure 31 shows contact angle versus time measured on a COC flow cytometry chip
which have been treated in accordance with the present invention, in particular
(a) an Ar/02plasma treated chip and (b) a COC substrate coated with TEOS. In
(a) the contact angle stay below 45° for 60 days and in (b) the contact angle
stay below 40° for 65 days.
figure 32 shows a comparison of the Zeta potential as a function of pH for different glass
surfaces to bare polymer surfaces and glass-like surfaces on polymer sub¬
strates. The IEP (isoelectric point), the pH below which the Zeta-potentials are
negative, can be seen to be shifted below pH 3 for the glass-like surfaces,
figure 33 shows a comparison of the Zeta potential as a function of pH for PMMA and
COP surfaces to an etched glass surface and a Si02 sol-gel covered surface, as
well as the effect of SDS on the different surfaces. While SDS strongly affects
the Zeta potentials on the bare PMMA and COP surfaces, the effect is much
weaker for the Si02 sol-gel covered substrate, similar to the behaviour ob
served for the etched glass surface,
figure 34 shows the chemical structure for nafion,
figure 35 shows a comparison of the Zeta potential as a function of pH for COP, COP
covered by a layer of nafion, and of a Si-Wafer covered by a 400 nm layer of
Si0 2.
Furthermore, reference is made to the following examples, which are given to illustrate, not to
limit the present invention:
Examples
In the following examples, the order in which the steps are listed typically is the order in
which these steps are performed for the experiment.
Example 1
COC substrate coated by evaporated Si0 2 film
(Figs. 1 - 3)
-Substrate: COP (Zeonor 1060R)
-Coating: 20 nm Si0 2 by thermal evaporation
- optical transparency is higher than 85% (see Fig. 1)
-water contact angle (after 12h): 5°, stabilizes at about 40° (see Fig. 2)
-the channel dimensions of the substrate are hardly modified by the coating procedure which
indicates that the channels are not totally filled or blocked with Si0 (see Fig. 3).
Example 2
PMMA substrates coated with TEOS films
(Figs. 4-10)
A general scheme for Si0 2 precursor coating is shown in Fig. 4, structural formulas for some
liquid precursors are given in Fig. 5.
- substrate: PMMA channel plate and well plate (Diakon CMG 302)
- Coating material: TEOS
- Topography: 0.5 nm roughness (see Fig. 6)
- Composition: XPS clearly shows Si picks at the substrate surface (Fig. 7)
- Bonding at 70 °C. The bonding scheme is shown in Fig 8. Bonding was successful with no
deformation of the channels and no leakage of fluids when filling the channels (see Fig. 9 and
Fig. 10).
Example 3
PMMA substrates coated with PHPS films
(Figs. 11-13)
- Substrate: PMMA channel plate and well plate (Diakon CMG 302)
- Coating material: Perhydropolysilazane (PHPS) (see Fig. 11)
- Post-treatment: NH4OH vapour
- Water contact angle: 15°
- Topography: 1.7 nm roughness (see Fig. 12), channels dimensions are not modified (see Fig.
13) and channels are not filled up or blocked.
- Bonding at 70°C. Bonding was successful.
Example 4
PMMA substrate coated with PTMSP film
(Fig. 14-15)
- Substrate: PMMA channel plate and well plate (Delpet 70NH )
- Coating material: Poly[l-(trimethylsilyl)-l-propyne] (PTMSP) PTMSP solution in Toluene
- Coating Procedure: Spraying,
- Characterisation: Contact angle: advancing: 107°, receding: 79°, the difference in the two
angles indicates a rough and porous surface; FTIR: PTMSP signals are clearly identified (see
Fig. 14); Profilometer: conduits dimensions are not modified, increased roughness is observed
with AFM (Fig 16); charging: PTMSP film can be charged (by biased-probe induced charg¬
ing) with water ions just like glass (see Fig. 15 a and b).
- Bonding at 70°C. Bonding was successful.
Example 5
PMMA substrate treated with Ar/0 plasma
(Fig. 16, 17)
- Substrate: PMMA channel plate and well plate, (Delpet 70NH)
- Treatment: Ar/0 2 Plasma
- Characterisation: Contact angle: 50° (advancing), receding: <10°; AFM: 25 nm rms rough¬
ness (see Fig. 16);
- Bonding at 70°C. Bonding was successful. An increased roughness and hydrophilicity mim¬
icking that of glass was achieved.
- Electrophoretic separation: successful DNA separation (see Fig 17a, and 17b separation on
glass chip for comparison). A chip in accordance with the present invention provides the same
electrophoretic separation of a DNA latter containing DNA molecules up to 7500 Dalton as
achieved with glass chip. The gel used for the electrophoretic separation is based on polyacrilamide
inl20mM Tris-Tricine (pH 7,7-8). Dissolved detergents are SDS and LDS as well as
fluorescent dye if staining occurs on chip.
Example 6
Wet coating of COP chip with Si0 sol-gel
(Fig. 18)
- Material: COP channel and well plate (Zeonor 1060R)
- Treatment: the substrate is exposed to chloroform vapor for few minutes before bonding.
- Bonding at 70°C. Bonding was successful.
- Wet-coating: Si0 2 sol-gel
- Electrophoretic separation: good protein separation (see Fig 18a, and 8b separation on glass
chip for comparison). A chip in accordance with the present invention provides the same elec¬
trophoretic separation of Bovine Serum Albumin (a protein) in different concentrations (e.g.
500 mg/ml, 1000 mg/ml, 2000 m pi ) as achieved with glass chip. Molecular weight marker
containing 6 proteins (29 kDa, 45 kDa, 66 kDa, 97 kDa, 116 kDa, 200 kDa). The gel used for
the electrophoretic separation is based on polyacrylamide inl20mM Tris-Tricine (pH 7,7-8).
Dissolved detergents are SDS and LDS as well as fluorescent dye if staining occurs on chip.
Example 7
COP substrate treated with Ar/0 2-plasuia
- Substrate: COP channel and well plate (Zeonor 1060R)
- Treatment: Ar/0 2 Plasma
- Bonding at 85°C and at constant pressure Bonding was successful. The obtained chip has no
bonding voids and no channel deformation (figure 19).
Example 8a
COP substrate treated with Ar plasma / UV-ozone
- Substrate: COP channel and well plate (Zeonor 1060R)
-Treatment: Ar plasma / UV-Ozone cleaner
- Bonding at 85°C and at constant pressure Bonding was successful (figure 20).
- Bonding force: 300N for 20s were applied. The samples did not detach.
Example 8b
PMMA Substrate treated with Ar-plasma /UV-Ozone
- Substrate: PMMA channel and well plate (PMMA Delpet 70NH)
- Treatment: Ar plasma/UV-Ozone
- Bonding at 85°C and at constant pressure Bonding was successful. Figure 2 1 shows an (a)
optical microscope image of a cross section of a COP bonded conduit formed by two sub¬
strates that have been treated with Ar plasma/UV-Ozone; there is no deformation of the struc¬
ture that can be seen in the optical microscope image, the dimensions of the channel/conduit
do not change upon treatment and bonding . Figure 2 1 (b) shows an electrophoretic separation
of DNA 7500 analyte obtained with a COP chip treated with Ar plasma/UV-Ozone.
Example 9
COP-Substrate treated with Si0 2 sol-gel
Substrate: COP channel and well plate (Zeonor 1060R)
Coating:
- Coating material: Si02 sol-gel
- Coating Procedure: Spraying
- Post treatment: 02-plasma
Bonding at 87°C Bonding was successful, the obtained chip has no bonding voids and no
channel deformation.
Example 10
PMMA-Substrate treated with Si02 sol-gel
Substrate: PMMA channel and well plate (PMMA Delpet 70NH)
Coating:
- Coating material: Si02 sol-gel
- Coating Procedure: Spraying
- Post treatment: 02-plasma
Bonding at 85°C was successful. The obtained chip has no bonding voids and no channel
deformation.
Example 11
Application of substrates according to the present invention for assay applications, such
as genome sequencing
Transferring assay chemistries which have been developed for glass substrate often requires
the addition of detergents to ensure the wetting of the hydrophobic plastic surface or to avoid
the sticking of proteins or other biomolecules on these hydrophobic surfaces. Adding such
detergents may negatively impact the performance of the assay, since such substances can
lead to denaturation of proteins or other biomolecules. Large protein molecules can easily
loose their functionality in the presence of detergents or other surface active substances. Pro¬
viding surfaces with glass like properties ensures easy transfer of such assays to plastic con¬
sumables. It should also be mentioned that Cells or cell fragments are most easily damaged or
destroyed by exposure to detergents or hydrophobic plastic surfaces. Any of the above exam¬
ples represent cases, where the protein or biomolecule, large protein assemblies or cells and
cell fragments are exposed to none native conditions in which they may behave very differ¬
ently from within their natural environment. A glass like coating in accordance with the present
invention with the right pH and ion concentration in the buffer can minimize such nega¬
tive influence
The substrates in accordance with the present invention can also be used to be applied in a
genome sequencing assay. Conventionally, such genome sequencing is performed in a silicon
chip having 50 million wells of a defined diameter and depth, wherein each well is filled with
a polystyrene bead decorated with DNA. Consequently, if such structure is to be manufac¬
tured using the substrates in accordance with the present invention, the same requirements
apply for the substrate in accordance with the present invention: The wells must be arranged
at a defined distance from each other (Figure 23); the substrate must have a defined smooth¬
ness, hydrophilicity and low cost. Typically, in one example, the smoothness of the substrate
is 1 mp on 1 mm, and the contact angle is between 20 and 50°.
The following treatment was performed:
COP treated with Ar/0 2 plasma
- Substrate: COP (Zeonor 1060R) with and without TiO2 filling material
- Treatment: Ar/O -plasma
Figure 24 shows contact angle measured in difference fields of an assay substrate that have
been treated in accordance with the present invention, repeated in different days after treat¬
ment. In particular a TiO2 filled COP substrate treated with Ar/O2 plasma shows a contact
angle below 65° after 114 days. The untreated TiO2 filled COP substrate has a contact angle
of 110-120°.
- COP treated with SiO2
- Substrates: COP (Zeonor 1060R) with and without TiO2 filling material,
- Coating: 20 nm SiO2 by thermal evaporation and sputtering
Profilometer measurements are shown in figure 25 which makes it clear that the differences
in height are rather small and are within a range of approximately 300 nm. At the same time,
the contact angle remains stable in various positions over a considerable amount of time, i.e.
up to 115 days, at least.
Figure 26 shows AFM topography scans of (a) molded wells in a plastic substrate, and (b), the
same plastic substrate after evaporation of 20 run Si02. Morphology and roughness of the
wells are not affected by the Si02 evaporation
Example 12
Application of substrates according to the present invention for flow cytometry
The substrates according to the present invention can also be used for flow cytometry applica¬
tions. In this respect, they need to fulfil the following requirements: there must be no cell ad¬
hesion, there must be a hydrophilicity with a contact angle between 20-50 degrees, and there
must be pressure durability. Furthermore, there must not be the possibility of air bubble for¬
mation during the loading of the chip.
Substrate treatment with Ar/02 plasma
:Substrates: COC half-channel plates (Topas 8007 X10)
Treatment: Ar/02 plasma
Bonding at 75°C at constant pressure was successful.
Figure 27 shows AFM images of the surface treated in accordance with this example accord¬
ing to the present invention. The roughness was increased after the Ar/02 treatment from 0.8
rms to 6 rms, showing that the same treatment can be apply on different polymeric material
obtaining the same surface morphology.
Figure 28 shows an XPS spectrum of (a) an untreated and a Ar/02 plasma treated (b) COC
flow cytometry chip sample to confirm that the treatment did not change the chemical compo¬
sition of the polymeric substrate.
Substrate treatment with TEOS
Substrates: COC half-channel plates (Topas 8007 X10)
Coating material: TEOS
Coating Procedure: Dipping
Post-treatment: 0 2 Plasma
Bonding at 76°C at constant pressure was successful.
Figure 29 shows substrate surfaces before and after the treatment according to the present
invention, and figure 30 shows the corresponding XPS measurements.
It can be seen that the surface morphology did not change (same rms on a 1 x 1 mhi2 ) while
the XPS confirmed the presence of Si02 on the surface of the treated sample.
The long-time stability of the treatments according to the present invention have also been
measured as can be seen in figure 30a and b which basically show that the contact angle of the
COC plates (Topas 8007 X10) stays below 50° for extended periods of time, thus proving that
the methods in accordance with the present invention will produce substrates that can be used
for commercial purposes and are also amenable to multi-use-applications.
Example 13
The substrates in accordance with the present invention can also be characterized to have
glass-like behaviour by measuring the respective zeta potential. As can be seen in figure 32,
the glass-like substrates in accordance with the present invention have zeta potential curves
that have the same shape and values as a glass surface. More specifically, the IEP (isoelectric
point), the pH below which the Zeta-potentials are negative, can be seen to be shifted below
pH 3 for the glass-like surfaces.
Furthermore, as can be seen in figure 33, the surfaces/substrates in accordance with the pre¬
sent invention have the same behaviour as glass, when in contact with SDS. More specifi¬
cally, the zeta potential is not much affected by the presents of SDS, whereas for substrates
not treated in accordance with the present invention, the influence of SDS is much bigger.
More specifically figure 33 shows a comparison of the Zeta potential as a function of pH for
PMMA and COP surfaces to an etched glass surface and a Si02 sol-gel covered surface, as
well as the effect of SDS on the different surfaces. While SDS strongly affects the Zeta poten¬
tials on the bare PMMA and COC surfaces, the effect is much weaker for the Si02 sol-gel
covered substrate, similar to the behaviour observed for the etched glass surface.
Example 14
One possibility for a surface treatment in accordance with the present invention is to treat the
polymeric substrate with nafion which is a sulfonated tetrafluoroethylene based fluoropolymer-
copolymer. It is a ionomer which forms ion-exchange membranes. It has a highly spe¬
cific conductance for protons in solution and allows a proton conduction due to the formation
of water channels. The structure of nafion and the mechanistic details of its behaviour can be
inspected in figure 34 which show the chemical structure of nafion as well as a scheme to
explain the behaviour of nafion in and towards water.
Figure 35 shows the zeta potential of a substrate in accordance with the present invention that
has been coated with nafion. The nafion coated substrate shows a very negative zeta potential
which is even more negative than glass.
This makes substrates in accordance with the present invention that have been treated with
nafion obtaining a glass-like surface.
The features of the present invention disclosed in the specification, the claims and/or in the
drawings may, both separately and in any combination thereof, be material for realizing the
invention in various forms thereof.
Claims
1. A polymeric substrate having a glass-like surface, in particular an etched-glass-like surface,
said glass-like surface, in particular said etched-glass-like surface mimicking the surface of
glass, in particular etched-glass in one or several of:
chemical content, chemical composition, chemical structure, homogeneity, roughness, mor¬
phology, in particular porosity, hydrophilicity, surface energy and adsorption affinity, surface
functionality, chemical and physical surface reactivity, zeta potential and surface charge.
2. The polymeric substrate according to claim 1, wherein said surface has been modified by
plasma treatment and/or reactive ion treatment for increased roughness and hydrophilicity
and/or is a thin film of silicon oxide and/or is a polymer thin film having increased intrinsic
roughness and/or intrinisic porosity and/or increased hydrophilicity.
3. The polymeric substrate according to any of claims 1-2, wherein said polymeric substrate is
a polymer of synthetic or natural origin, preferably injection-mo ldable polymers, more pref¬
erably one of the material classes containing: polyolefines, polyethers, polyesters, polyamides,
polyimides, polyvinylchlorides, polyacrylates; including their modifications, derivates,
derivatives and copolymers; more specifically one of the list containing acrylnitril-butadienstyrole
(ABS), cyclo-olefin-polymers and copolymers (COC/COP), Polymethylenemethacrylate
(PMMA), Polycarbonate (PC), Polystyrole (PS), Polypropylene (PP), Polyvinylchloride
(PVC), Polyamide (PA), Polyethylene (PE), Polyethylene-terephthalate (PET),
Polytetrafluor-ethylene (PTFE), Polyoxymethylene (POM), Thermoplastic elastomers (TPE),
thermoplastic polyurethane (TPU), Polyimide (PI), Polyether-ether-ketone (PEEK), Polylactic
acid (PLA), polymethylpentene (PMP), and derivatives thereof, wherein said polymer is op¬
tionally filled with an inorganic material such as carbon black,oxides such as Si0 2, A 120 3,
Ti0 2, Zr0 2, Fe20 3, in particular metal oxides, and semiconductors such as ZnS, CdS, CdSe.
4. A method of providing a polymeric substrate with a glass-like surface, in particular an
etched-glass-like surface, said method comprising the following step:
a) providing a polymeric substrate which, preferably, is made of a polymeric substrate mate¬
rial as defined in claim 3;
and one of the following steps:
bl) coating a surface of said polymeric substrate with SiOx; x being in the range of from 1 to
2;
bl) coating a surface of said polymeric substrate with a SiCvprecursor and converting said
SiOx-precursor into SiOx, x being in the range of from 1 to 2;
b3) endowing a surface of said polymeric substrate with a glass-like surface morphology, in
particular an etched glass-like surface morphology, by
coating a surface of said polymeric substrate with a coating material having increased
roughness in comparison to the uncoated surface of said polymeric substrate and/or in¬
creased hydrophilicity, said coating material preferably being a polymer, and/or
by plasma-treating and/or reactive ion etching (RIE) and/or by treatment with a UVOzone
cleaner of a surface of said polymeric substrate or said coated polymeric sub¬
strate, preferably using argon, oxygen, H20 , H2, fluorinated methane gases, such as
CF , CHF3, CH2F2, or a mixture of or a sequential use of any of the foregoing, to in¬
duce roughness and hydrophilicity on said surface;
b4) a combination of bl), bl) and b3) in any order.
5. The method according to claim 4, wherein step bl), b2) and/or b3) is performed
by physical vapor deposition methods, such as:
- thermal deposition (physical vapor deposition)
- electron-beam (E-gun) deposition
- sputtering,
- chemical vapor deposition (CVD),
- film growth on the surface, such as electroless plating or electrochemical deposition,
- spray coating
- dip coating
- gas-phase deposition
- roll-to-roll deposition,
- screen printing,
- doctor blading,
- wet coating,
- dynamic coating,
- CVD,
or a combination of several of the foregoing.
6. The method according to any of claims 4-5, wherein the converting in step b2) is performed
by one of:
- annealing, preferably thermal annealing
- irradiating with electromagnetic radiation, such as infrared or UV, preferably UV
- treatment in solution with water or base or acid, or a combination of water and base, or a
combination of water and acid.
7. The method according to any of claims 4-6, wherein said SiO -precursor is selected from
the group comprising
i) alkoxy- or alkyl-chlorosilane, S1X4, trisiloxane compound S13O2X6, X being, independently,
at each occurrence OR or halogen, R being alkyl, alkyl being preferably Ci-C 20-alkyl, pref¬
erably ethyl or methyl;
ii) polysilazane, such as perhydropolysilazane, -[Si(H)2-N(H)-] n, n being selected from 3 to
10000, or polyorganosilazane -[Si (R)2-N(R)-] n, R being alkyl, alkyl being preferably Ci-C 20-
alkyl, n being selected from 3 to 100 000;
iii)a sol-gel containing SiOx particles, said particles preferably having a diameter of about 1
nm to 10 mhi, preferably 10 nm to 100 nm, suspended in a solvent-based matrix , the solvent
being preferably an alcohol, preferably ethanol.
8. The method according to any of claims 4-7, wherein, in steps bl) - b3), together with said
SiOx, SiOx-precursor or said polymer, additional materials are coated on said surface of said
polymeric substrate, said additional material being selected from S12N3, AI2O3, B20 3, T1O2,
Na20 , CaO, 20 , S0 3, MgO, Fe20 3, SiO x (x being < 2).
9. The method according to any of claims 4-8, wherein said coating material, in particular said
polymer, having increased roughness and/or intrinsic porosity is selected from polytetrafluoroethylenes
(Teflon), such as 2,2-bistrifluoromethyl-4,5-difluoro-l,3-
dioxoletetrafluoroethylene copolymer (designated Teflon AF 2400 by Du Pont), nafion, polysulfones,
poly(oxy-2,6-dimethyl-l,4-phenylene), and the group of substituted acetylene poly¬
mers, like poly [l-(trimethylsilyl)-l-propyne] (PTMSP), poly[l-(trimethylgermyl)-l-propyne],
poly(4-methyl-2-pentyne), poly [1-phenyl-2-(/?-trimethylsilylphenyl)acetylene] , and
poly[2,4,5-tris(trifluoromethyl)-phenylacetylene].
10. The method according to any of claims 4-9, wherein said method further comprises a step
c) post-treating said substrate by one or several of
- drying,
- water treatment,
- tempering,
- sintering,
- solvent treatment,
- plasma treatment,
- reactive ion etching,
- treatment with UV-Ozone cleaner,
and any combination of the foregoing.
11. The method according to any of claims 4-10, wherein said method is performed at least on
a first polymeric substrate made of a material, as defined in claim 3, wherein said first poly¬
meric substrate thereby is provided with at least a first glass-like surface, in particular a first
etched-glass-like surface, said method further comprising the bonding step:
bonding said first substrate to a second substrate by bringing said first glass-like surface, in
particular etched-glass-like surface in contact with a surface of a second substrate, by pressing
said first and second substrate together, preferably by a pressure in the range of from 0.2
N/mm 2 to 5 N/mm 2, more preferably in the range of from 0.5 N/mm 2 to lN/mm 2, for a time in
the range of from 10 s to 600 s, more preferably in the range of from 30 s to 120 s and by ex¬
posing said first and second substrate to a temperature in the range of from 40°C to 200°C,
preferably 60°C to 120°C, and/or exposing said first and second substrate to a vapor of sol¬
vent immediately before pressing to increase their bonding to each other.
12. The method according to claim 11, wherein said first polymeric substrate is a solid sub¬
strate, and wherein said second substrate is either a solid substrate or a flexible foil.
13. The method according to any of claims 11-12, wherein said second substrate is a poly¬
meric substrate made of a material as defined in claim 3, or said second substrate is made of a
material selected from glass, quartz, silicon nitride and silicon oxide, or, if said second sub¬
strate is a flexible foil, said second substrate is made of a plastic material such as polyolefines,
polyethers, polyesters, polyamides, polyimides, polyvinylchlorides, polyacrylates; including
their modifications, derivates, derivatives and copolymers; more specifically one of the list
containing acrylntiril-butadien-styrole (ABS), cyclo-olefin-polymers and copolymers
(COC/COP), Polymethylene-methacrylate (PMMA), Polycarbonate (PC), Polystyrole (PS),
Polypropylene (PP), Polyvinylchloride (PVC), Polyamide (PA), Polyethylene (PE), Polyethylene-
terephthalate (PET), Polytetrafluor-ethylene (PTFE), Polyoxymethylene (POM), Ther¬
moplastic elastomers (TPE), thermoplastic polyurethane (TPU), Polyimide (PI), Polyetherether-
ketone (PEEK), Polylactic acid (PLA), polymethylpentene (PMP) and derivatives
thereof, wherein said polymer is optionally filled with an inorganic material such as carbon
black, oxides, such as Si0 2, A 1 0 3, Ti0 2, Zr0 2, Fe20 3, in particular metal oxides, and semi¬
conductors, such as ZnS, CdS, CdSe .
14. The method according to any of claims 11-13, wherein one or both of said first substrate
and said second substrate has a channel or groove or recess or hole in it, which, preferably,
forms a conduit at the interface between said first and second glass-like surfaces, wherein
more preferably, said conduit extends from one edge to another edge of said first or second
substrate or from one hole of one of the substrates to another hole of one of said substrates,
thus allowing the flow-through of a liquid through said conduit.
15. The method according to any of claims 11-14, wherein the method of any of claims 4-10
is performed on said first polymeric substrate and on at least a second polymeric substrate
made of a material as defined in claim 3 and, optionally, on a third and/or further polymeric
substrates, each being made of a material as defined in claim 3, wherein said first and second
polymeric substrate and, if present, said third and further polymeric substrates are subse¬
quently bonded to each other by the bonding step as defined in claim 11.
16. The method according to claim 15, wherein one, two or more of said polymeric substrates
have a channel or groove or recess or hole, which forms a conduit at the interface between
said substrates, wherein, preferably, said conduit extends from one edge to another edge of
said substrate(s) or from one hole of one of the substrates to another hole of one of said sub¬
strates, thus allowing the flow-through of a liquid through said conduit.
17. A method of manufacturing a chip, preferably, a microfiuidic chip, said method compris¬
ing:
a) providing at least a first polymeric substrate, made of a material as defined in claim 3, and
a second substrate, said second substrate being a substrate as defined in claim 12 and made of
a material as defined in claim 13, wherein at least one of said first and said second substrate
have a channel or recess or groove or hole in it; and either
bl) performing the method according to any of claims 4-10 on at least said first substrate,
preferably on both said first and second substrate, and subsequently performing the bonding
step according to claim 11 thereon; or
b2) bringing said first and said second substrate in contact with each other and bonding them
with each other by the bonding step of claim 11 to form an assembly of said first and second
substrate, and subsequently, performing the method according to any of claims 4-10 on said
assembly, in particular on the conduit(s) which is (are) formed inside said assembly.
18. The method according to claim 17, wherein step a) includes providing a plurality of sub¬
strates, as defined in any of claims 12 and 13, and steps bl) or b2) are performed on said plu¬
rality of substrates, thus resulting in a chip comprising a plurality of substrates which are
stacked on one another.
19. A polymeric substrate produced by the method according to any of claims 4-10.
20. A chip made of two substrates, at least one of which, preferably at least two of which are
as defined in any of claims 1-3 and 19, said chip having at least one conduit going through
said chip, said conduit preferably having a smallest dimension being in the range < 500 m ,
preferably < 200 m .
21. The chip according to claim 20 made of one substrate as defined in any of claims 1-3 and
19 and either
- a) another substrate as defined in any of claims 1-3 and 19,
- or b) another substrate which is a flexible foil as defined in any of claims 12 and 13,
- or c) another substrate which is made of a material selected from glass, quartz, silicon ni¬
tride, and silicon oxide,
- or d) a plurality of substrates, wherein said plurality of substrates is composed of a plurality
of substrates of the type defined in a) or b) or c), or is composed of a plurality of substrates of
any combination of the types defined in a) - c).
22. The chip according to any of claims 20-21, wherein said at least one conduit is filled with
a matrix suitable for the analysis and/or detection and/or separation and/or transport of analytes,
preferably of biological cells, or biological macromolecules and/or their respective
mono/oligomers, such as nucleic acids, nucleotides, proteins, peptides, amino acids, carbohy¬
drates, polysaccharides, oligosaccharides, monosaccharides, lipids, fats, and fatty acids.
23. The chip according to claim 22, wherein said matrix is a gas phase, a liquid, a dispersion
of solid particles in a liquid, or a gel, preferably a polyacrylamide gel or an agarose gel or a
starch gel or a sucrose gel.
24. A chip produced by the method according to any of claims 17-18, in particular the chip
according to any of claims 20-23.
25. A kit for manufacturing a chip by the method of any of claims 17-18, said chip being de¬
fined as in any of claims 20-24, said kit comprising at least a first substrate and a second sub¬
strate,
wherein at least one of said first and second substrate has a channel or recess or groove or
hole in it, said kit further comprising one or several agents to perform the method according
to any of claims 4-10 on said substrates.
26. The kit according to claim 25, wherein said first and second substrate is made of a mate¬
rial selected from polymers of synthetic or natural origin, preferably injection-moldable
polymers, more preferably one of the material classes containing: polyolefines, polyethers,
polyesters, polyamides, polyimides, polyvinylchlorides, polyacrylates; including their modifi¬
cations, derivates, derivatives and copolymers; more specifically one of the list containing
acrylntiril-butadien-styrole (ABS), cyclo-olefin-polymers and copolymers (COC/COP), Polymethylene-
methacrylate (PMMA), Polycarbonate (PC), Polystyrole (PS), Polypropylene
(PP), Polyvinylchloride (PVC), Polyamide (PA), Polyethylene (PE), Polyethyleneterephtalate
(PET), Polytetrafluor-ethylene (PTFE), Polyoxymethylene (POM), Thermoplas¬
tic elastomers (TPE), thermoplastic polyurethane (TPU), Polyimide (PI), Polyether-etherketone
(PEEK), Polylactic acid (PLA), polymethylpentene (PMP), and derivatives thereof
wherein said polymer is optionally filled with an inorganic material such as carbon
black,oxides, such as Si0 2, AI2O3, Ti0 2, Zr0 2, Fe20 3, in particular metal oxides, and semi¬
conductors such as ZnS, CdS, CdSe ..
27. A kit for the analysis and/or detection and/or separation and/or transport of analytes, pref¬
erably of biological macromolecules and their respective mono/oligomers, said kit compris¬
ing:
the chip according to any of claims 20-24, and, optionally, one or several agents, such as e.g.
buffers, necessary for the analysis and/or detection and/or separation and/or transport of ana¬
lytes.
28. Use of a polymeric substrate according to any of claims 1-3, 19, or of a chip according to
any of claims 20-24 in a method of analysing, detecting, separating and/or transporting ana¬
lytes, preferably biological cells, or biological macromolecules and/or their respective
mono/oligomers, in particular in a method of electrophoresis, a method of sequencing, an as¬
say method for the qualitative or qualitative detection of an analyte or its activity, or a method
of flow cytometry.
29. A method for analysing, detecting, separating and/or transporting analytes, preferably bio¬
logical macromolecules and/or their respective mono/oligomers, comprising:
- providing, in any order, a chip according to any of claims 22-23 or a polymeric substrate
according to any of claims 1-3, 19, and a sample containing analyte(s) to be analysed, sepa¬
rated and/or transported, said polymeric substrate having a matrix as defined in claim 22 at¬
tached,
- applying said sample to said matrix,
- applying a voltage to said matrix, said voltage being sufficient to cause the migration or dif¬
fusion or flow of the analyte(s) through said matrix,
- irradiating or ablating or desorbing or ionising the analyte(s), and, optionally, further
- analysing the analyte(s).
30. Use of a substrate according to any of claims 1-3 and 19 for manufacturing a chip, in par¬
ticular a microfluidic chip, or plastic substrate for the analysis and/or detection and/or separa¬
tion and/or transport of analytes.